The present invention relates to immunoassays for non-collagen cartilage proteins and their fragments in biological samples such as a body fluids. Such proteins and protein fragments may serve as an index of joint disease.
Rheumatoid arthritis (RA) is a severe chronic and progressive disease affecting approximately 1% of the population in both the industrialised and the developing world (Harris 1993). Although both environmental, genetic and developmental factors have been implicated in the aetiology of RA, it is now generally accepted that RA is an autoimmune disease. Osteoarthritis (OA) is a chronic disease affecting more than 8% of the population in the industrialised world. This disease also affects the articular cartilage of joints, and although an immune component has been observed as part of the disease pathophysiology, OA is not viewed as an autoimmune disease.
The major clinical manifestation of RA as well as OA is an abnormal and degraded cartilage. However, until now it has been difficult to directly assess the ongoing cartilage destruction in arthritis patients, because specific markers for this process have not been available in the clinical practice (Møller 1998). At clinical diagnosis of RA, the patients are scored according to the disease symptoms and function impairment such as pain, and mobility problems caused by the joint destruction. Even though a number of standardised rating systems have been introduced, it is difficult to quantify these parameters (Stucki et al 1997). Other markers used for assessment of RA patients, such as C-reactive protein and Rheumatoid factors are specific for the inflammatory process involved in the disease, but are not directly related to the level of cartilage destruction and they are not specific for RA (Wollheim 1996). At present one of the best ways to obtain information about the status of the (individual) joints in arthritis patients is radiological examinations.
Measurement of metabolites, such as hyaluronates and aggrecan fragments arising from destruction of the joints affected by the disease have been reported (Møller 1998, Wollheim 1996). The clinical usefulness of these markers, however, remains to be proven.
This invention is based upon a new approach for identifying markers of cartilage degradation, and for development of diagnostic and prognostic assays for monitoring joint diseases. We have shown that specific components of articular cartilage are prone to isomerisation and/or optical inversion (FIG. 1) and we have identified specific isomerisation/optical inversion prone sites in several cartilage proteins. We have also demonstrated that isomerised and/or optically inverted fragments of cartilage protein are found in circulation, and that measurements of such fragments provide an index of joint cartilage degradation.
Aspartic acid and asparagine (Asx) and glutamic acid and glutamine (Glx) residues will in some susceptible proteins undergo a spontaneous re-arrangement where the normal peptide bond between the Asx and Glx residue and the adjacent residue is transferred from the normal α-carboxyl group to the β-carboxyl group (γ-carboxyl group for the Glx residues) of the side chain (Clarke 1987). The isomerisation reaction proceeds via an imide intermediate, which upon spontaneous hydrolysis may result in one of four forms: the normally occurring αL, the isoform βL, or the two optically inverted forms αD and βD as outlined in the following reaction scheme for aspartic acid-glycine. (The reaction occurs analogously for other susceptible Asx and Glx containing sequences):

The attack by the peptide backbone nitrogen on the side chain carbonyl group of an adjacent aspartyl residue can result in the formation of an imide ring, (A→B). The imide ring is prone to hydrolysis and optical inversion yielding peptides and isopeptides in both the D and L configurations. Optical inversion proceeds through a carbanion intermediate (D, E and F) either through direct proton abstraction (ADG or CFI) or via the imide pathway (BEH). Throughout the figure the peptide backbone is shown as a bold line.
However, in order for cyclic imide formation (and isomerisation/optical inversion) to occur, the three dimensional structure surrounding the Asx or Glx residues must have an optimal conformation and sufficient flexibility (Geiger and Clarke 1987).
Studies indicate that optical inversion of Asx residues in peptides and proteins primarily proceeds through the imide pathway (BEH) (Geiger and Clarke 1987, Radkiewics et al. 1996). However, other pathways such as direct proton abstraction or imino-δ-lactone formation may also contribute to optical inversion (Radkiewics et al. 1996). These pathways are however assumed to be of less importance (Geiger and Clarke 1987, Radkiewics et al 1996).
Isomerisation and optical inversion via the imide intermediate as outlined above is a spontaneous reaction occurring with a slow rate under physiological conditions (Geiger & Clarke 1987, Fledelius et al. 1997). As for all chemical reactions, the reaction speed can be accelerated by increasing temperature.
The introduction of such structural changes in a protein or peptide has profound effects on its function, stability and physical and chemical properties. Among other properties, the proteolytic degradation of proteins and peptides containing isomerised peptide bonds and/or optically inverted amino acids is significantly reduced compared to proteins and peptides composed exclusively of αL amino acids (Rafferty et al. 1988) Thus, protein fragments containing such modifications are not degraded to the same extent during normal tissue turnover (Van Regenmortel & Muller 1998) and they are much more likely to be present in circulation in measurable concentrations. Furthermore, by measuring proteins or protein fragments containing isomerised and/or optically inverted peptide linkages, newly synthesised molecules will not contribute to the measurements, and it will thus reflect ongoing degradation processes.
In WO96/30765, we disclosed that isomerised fragments of Type I collagen provided an improved index of bone resorption. It was further disclosed that Type II collagen (as found in cartilage) also contained potential isomerisation sites.
We have demonstrated that articular cartilage, a tissue with a very slow metabolism, contain non-collagen proteins which are subject to isomerisation and optical inversion and we have demonstrated that measurement of these proteins, or fragments thereof, can provide an index of joint cartilage degradation of diagnostic potential for assessing and monitoring joint diseases such as RA and OA.